Chromatographic Media and Chromatographic Equipment Storage Solutions and Use Thereof

ABSTRACT

Buffered antimicrobial solutions of benzyl alcohol and ethanol and their use for short or long term storage of chromatographic solids.

FIELD OF THE INVENTION

This invention relates to a composition that is stable and suitable forstorage of chromatographic media and chromatographic equipment forextended periods of time, e.g., for up to about two years or more. Theinvention also relates to methods of preparation and use of novelsolutions for making liquid-solid suspensions that inhibit bacterialgrowth and kill bacteria. The invention also discloses composition orsolutions for making liquid-solid suspensions and storing suchsuspensions.

BACKGROUND TO THE INVENTION

Chromatographic analysis is one of the commonly used analyticaltechniques for determining the properties, components and/orcharacteristics of various materials. However, although chromatographicanalysis has been widely adopted one problem that has arisen is theproblem of the media and equipment utilized in such chromatographicanalysis becoming impure or infected with bacterial matter, particularlywhen the chromatographic media and chromatographic equipment aresubjected to storage, particularly long term storage when not beingutilized. The prior art has not offered any effective solution to theproblem of safe and effective storage of chromatographic media andchromatographic equipment, especially for long term storage.

Various solutions such as phosphoric acid (0.01-0.5N), hydrochloric acid(0.1-0.5N), NaOH (0.1-2N) and alcohol (ethanol or isopropanol at minimum20% v/v in water) are widely used for bacterial inhibition. However,these solutions are highly acidic or basic or carry some environmentaland handling risks and are not compatible with chromatographic media andchromatographic equipment. Under current practice, if sodium hydroxide(0.1 M-0.5 M) or acids (0.1 M-0.5 M) are used, they need to be firstneutralized either by using acid or base, respectively, followed bywashing with sufficient water to remove excess salt. If 20% or higherconcentrations of alcohol (ethanol or isopropyl alcohol) are needed forbactericidal effect, it requires proper organic handling and disposal.In addition to the above solutions, some solution compositionscontaining buffer of pH 5.0 or lower and 1.0% benzyl alcohol have beenused for storage of some chromatographic media. However, due to reducedstability of the chromatographic media at low pH, low pH formulationsare not suitable for long-term storage. Benzyl alcohol is anantimicrobial preservative against gram positive bacteria and is used incosmetics, foods and a wide range of pharmaceutical formulationsalthough it possesses only modest bactericidal properties with optimalactivity known to occur below pH 5.0 as noted in Karabit, M. S.,Juneskans, O. T. and Lundgren, P. Journal of Clinical and HospitalPharmacy (1986) 11, 281-289; and R. C. Rowe, P. J. Sheskey and P. J.Weller, Handbook of Pharmaceutical Excipients, 4^(th) edition AmericanPharmaceutical Association, (2003) 53-55. In addition, the activity ofbenzyl alcohol has been shown to be reduced by incompatibilities withsome packaging materials such as methylcellulose, polyethylene,polystyrene, natural rubber, neoprene and butyl rubber and the like, asstated in R. C. Rowe, P. J. Sheskey and P. J. Weller, Handbook ofPharmaceutical Excipients, 4^(th) edition, American PharmaceuticalAssociation (2003), 53-55 and M. S. Roberts et. al, Int. J. Pharm.(1979) 2, 295-306].

Thus, there remains a clear need for an acceptable composition orsolution that will allow for safe and effective storage ofchromatographic media and chromatographic equipment with no oressentially minimalized risk of bacterial infection of the media orequipment occurring. There is especially a need for a composition orsolution that will allow for safe and effective storage ofchromatographic media and chromatographic equipment for an extendedperiod of time, e.g., about two or more years time, with no oressentially minimalized risk of bacterial infection of the media orequipment occurring.

SUMMARY OF THE INVENTION

It has been unexpectedly discovered that by employing compositions ofantimicrobial solutions one can provide solutions with essentiallyneutral pH, generally from about pH 5.5 to 7.5, preferably a pH of about6.0 to about 7.5, that are compatible with chromatographic solids, suchas chromatographic supports or media and chromatographic equipment, andthat such solutions maintain microbial killing and inhibitionproperties. The compositions or solutions of this invention comprisebuffered anti-microbial solutions of about 1.5% to about 4% benzylalcohol, about 0.5% to about 4% ethyl alcohol and generally from about92% to about 98% of about 50 mM to about 200 mM buffer to providesolutions at pH of about 5.5 to about 7.5, wherein the percentages arepercent by weight. In such solutions chromatographic solids, such asmedia, supports and equipment, can be safely stored for short orextended periods of time without undue risk of bacterial infection ofthe media, support or equipment occurring. The solutions of thisinvention can kill bacteria and inhibit bacterial growth in the presenceof solids when chromatographic media, supports or equipment are storedin these solutions for short or long periods of time. Another feature ofthis invention is that it provides such solutions that are neitherhighly basic nor acidic and that do not contain large (high percentage)of organic solvent. Additionally, the solutions of this invention areinert to commonly used chromatographic media and equipment materials ofconstruction. Moreover, the solutions of this invention are of lowtoxicity and are environmentally friendly.

A preferred composition includes 100 mM sodium phosphate-acetate bufferto provide a solution of pH 6.0-pH 7.0 containing 2% benzyl alcohol and2% ethanol. This solution can be used for storage of chromatographicmedia based on silica or polymeric beads. The liquid-solid suspensionmade using the chromatographic media and said solution is shown to killE. coli and C. albicans within 2 hours and A. niger within 24 hourswhile maintaining solutions bacteria free for extended times. Inaddition, this solution is also shown to kill various microbes presenton the solid support such as chromatographic media and maintain itbacteria free over the long term. Since this solution is highly inertand nontoxic, it permits usual operations such as packingchromatographic columns or equipment cleaning. In addition, the solutioncomponents can be easily removed from surfaces such as chromatographicmedia or equipment surfaces by washing with water, other buffers ordilute acids such as acetic acid (1.0%) which are normally used in thechromatographic operations.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

A purpose of the invention is to prepare solutions that can killbacteria and inhibit bacterial growth in presence of chromatographicsolids when the solution or chromatographic solids are stored in thissolution for short to long term. Another purpose of the invention is toprovide a solution that is not highly acidic or basic, does not containhigh amounts of organic solvent, is inert, i.e., does not damagecommonly used chromatographic media and equipment materials ofconstruction, is of low toxicity and environmentally friendly.

The invention relates to providing novel solutions for makingliquid-solid suspensions that inhibit microbial growth and killmicrobial flora. More particularly this invention relates to storage ofchromatographic solids, particularly chromatographic media in containersor chromatographic process columns. The invention process comprisesproviding antimicrobial buffer solution containing about 1.5 to about 4%benzyl alcohol, about 0.5 to about 4% ethyl alcohol, about 92 to about98% about 50 mM to about 200 mM buffer solution of pH of about 5.5 toabout 7.5. Another aspect of the invention comprises storing ofchromatographic solids for short or long periods of time in suchantimicrobial buffer solutions. This is accomplished, for example, bymixing about 1 part of chromatographic media with about 1-4 parts ofantimicrobial buffer solution in a container or another aspect of theinvention is storing the antimicrobial slurry of the chromatographicmedia in the antimicrobial buffer solution in a closed container or in achromatographic column until next use.

The buffered antimicrobial solution can be made by mixing buffersolution with ethanol and benzyl alcohol. In a preferred embodiment, thebenzyl alcohol is first mixed with the ethanol and then adding thebuffer to provide the desired pH of from about 5.5 to 7.5. Examples ofvarious suitable buffering agents that may be employed include, but arenot limited to, sodium citrate, sodium acetate, sodium phosphate,triethanolamine, tris(hydromethyl)aminomethane (TRIS), HEPES(N-(2-hydroxyethyl)piperizine-N′-(2-ethanesulphonate)), and MES(4-morpholineethanesulphonate).

The preferred antimicrobial composition of this invention is about 2%ethanol, about 2% benzyl alcohol and about 96% about 100 mM sodiumphosphate (dibasic) titrated to pH 6.0 with acetic acid in water. Theantimicrobial solution or chromatographic solids-buffered solutionslurry stored in polypropylene containers is shown to maintain itsantimicrobial efficacy and chemical composition for at least 2 years.

The antimicrobial solution and its use for storage of chromatographicmedia render it very suitable for industrial use from small to largescale. The antimicrobial solution of this invention uses benzyl alcoholthat is generally regarded as safe (GRAS) along with less than 4% ethylalcohol which makes it easy to handle and that is readily disposed. Inaddition, the benzyl alcohol can easily be removed from thechromatographic media by either washing with generally usedchromatographic buffers such as in the concentration range of about 10mM to about 1 M in the pH range of 3.0 to 9.0. For example TRIS, HEPES,MES, sodium acetate, sodium citrate and sodium phosphate can be used.Alternatively, dilute acetic acid (0.01 M to 0.5 M) can also be used forremoval of antimicrobial solution and components from thechromatographic solids such as chromatographic media.

The materials and methods used in this disclosure of the invention areas follows:

Test Microbes.

Microbes used in the various assays were obtained from American TypeCulture Collection (ATCC), 10810 University Blvd., Manassas, Va.20110-2209. The following microbes were obtained: Candida albicans (ATCC#10231), Aspergillus niger (ATCC #16404), Staphylococcus aureus (ATCC#6538) and Escherichia coli (ATCC #8739). Cultures of C. albicans weremaintained on 2% Yeast Malt Agar (YMA) (Sigma Chemical Co. # Y3127).Cultures of A. niger were maintained on 2% Potato Dextrose Agar (PDA)(Sigma Chemical Co. # P2182). Cultures of S. aureus and E. coli weremaintained on 2% Nutrient Agar (NA) (Sigma Chemical Co. # N0394). Allcultures were kept at room temperature or at 5-10° C. until needed andwere periodically (approximately weekly) streaked onto fresh sterileplates of media.

Antimicrobial Effect Determination by Colony-Forming Unit Method.

A single colony of any given microbe was picked with a sterile loop andtransferred to sterile water or 1× phosphate buffered saline and dilutedto approx. 10,000 colony-forming units (CFU's) per mL. To 5 mL of a testantimicrobial solution (or sterile water or 0.1-1.0× phosphate bufferedsaline to serve as a CONTROL) was added 0.1 mL of approx. 10,000 CFU/mLculture and mixed briefly by gentle vortexing. At various times, 0.1-1.0mL of inoculated antimicrobial solution was transferred to the center ofan empty sterile plate and 15 mL of sterile media (agar) held at 45-50°C. was added and thoroughly mixed. The media added for each respectivemicrobe was the same as that used for maintaining the cultures. Plateswere held at room temperature for 20-45 minutes then transferred to anincubator maintained at 34-37° C. where they were held for 4 days.Periodically, the plates were examined for growth and colonies werecounted. Typically, colonies were apparent in 24 hours and counted at24, 48 and 72 hours. Usually the counts at 48 and 72 hours wereidentical. As a 2^(nd) CONTROL, a sample of test antimicrobial solution(0.1-1.0 mL) with no microbes added was added to the center of a sterileplate and 0.1 mL of the CONTROL culture of the microbe in question at1,000-2,000 CFU's/mL added to the plate at a distance of at least 2 cmfrom the test sample. As before, 15 mL of sterile media (agar) of choicefor that microbe held at 45-50° C. was added and thoroughly mixed. Thisserved to determine if the presence of the test components wereinhibiting growth on the plate rather than killing the microbes in theinoculated antimicrobial solution.

Examples Example 1 Various Buffering Compositions

Buffers of various types and concentrations (0.02-1.0 M) were titratedto pH's of 5.0-7.5 with appropriate acids or bases then ethanol andbenzyl alcohols were added to give 2% each. Representative buffers weresodium acetate (acetic acid titrated with sodium hydroxide),acetate-triethanolamine (acetic acid titrated with triethanolamine),sodium phosphate-acetic acid (sodium phosphate, dibasic, titrated withacetic acid) and sodium phosphate (sodium phosphate, monobasic, titratedwith sodium hydroxide). Typically, the buffering agent of choice wasmade up at 1.1-1.3 times the desired concentration and titrated to thedesired pH then diluted with water to give the desired concentration.The pH's remained the same as the undiluted stock. To a 100 mLvolumetric flask was added 2.0±0.02 grams benzyl alcohol and 2±0.01grams ethanol, and then the buffering solution was added. On someoccasions, the final solution was filtered through a sterile 0.2 micronfilter into a sterile flask to render it assuredly sterile. This wasfound to not be generally necessary as several trials with solutionscontaining 1% or greater concentrations of benzyl alcohol and othersterilants (with or without ethanol present) indicated that 24 hourincubation was sufficient to render the solution free of endogenousmicrobes. The test was begun by transferring 5 mL of the test solutioninto a sterile glass tube and adding 0.1 mL of microbial culture, mixingwell and maintaining at room temperature (20-23° C.) for an appropriateamount of time of about 1 to 24 hours to allow bacterial growth orkilling then transferring 0.1-1.0 mL to sterile plates. To each of theplates was then added 15 mL of appropriate bacterial growth media at45-55° C. and the media mixed well with the test culture. Colonyformation was determined after appropriate incubation at 34-37° C. for48-72 hours. It was found that the type of buffer seemed to have littleinfluence on the rate of killing microbes but the concentration and pHwas often important. Concentrations required to give substantialincrease in killing rates were generally over 0.1 M. Also, more microbeswere killed at pH's below 5.5 than at pH's higher than 5.5.

Example 2 Antimicrobial and Killing Using Antimicrobial Solution at pH4.0-7.5

Buffer compositions were made by titrating 0.025 M sodium phosphate,dibasic, with glacial acetic acid to various pH's in 0.5 pH unitincrements. To a 100 mL volumetric flask was added 2.0±0.02 grams benzylalcohol and 2±0.01 grams ethanol, then filled to the mark with 0.025 Msodium phosphate-acetic acid at a given pH. The pH remained the same asbuffer alone ±0.09 pH units. These mixtures were left at roomtemperature for at least 24 hours to allow elimination of endogenousmicrobes, which were typically 0, anyway. These solutions were tested byadding active cultures of microbes to 5 mL of test solution andincubating for an appropriate amount of time at room temperature.Samples of the test mixture were periodically plated with theappropriate media for the microbe in question and grown for 72-96 hoursat 33-37° C. Although the killing rate at pH 4.5 and below wasconsiderably faster than at pH 5.5-7.5, the pH 4.5 acidic solution wasnot acceptably stable and is detrimental to the chromatographic mediawhereas the killing rate at pH 5.5-6.5 was acceptable, requiring about24 hours to eliminate >95% of the active A. niger cells and less than 2and 4 hours to kill 100% of the active E. coli and C. albicans cells,respectively, yet was not detrimental to the chromatographic media andsuch solutions of about pH 5.5 to 6.5 were stable.

Example 3 Antimicrobial Effect in Slurry of Chromatographic Media

Buffer compositions were made by titrating 0.025 M sodium phosphate,dibasic, with glacial acetic acid to various pH's. To a 100 mLvolumetric flask was added either 1.5±0.2 or 2.0±0.02 grams benzylalcohol and either 1.5±0.2 or 2±0.01 grams ethanol, then filled to themark with 0.025 M sodium phosphate-acetic acid at a given pH. The pHremained the same as buffer alone ±0.09 pH units. These mixtures wereleft at room temperature for at least 24 hours to allow elimination ofendogenous microbes, which were typically 0, anyway. Meanwhile,approximately 3.1 packed mL of Protein A—silica media that had beenstored in 25 mM sodium phosphate-acetic acid, pH 6, containing 2% benzylalcohol and 2% ethanol at room temperature for 48 hours was centrifugedbriefly and the supematant siphoned off. The packed pellet was suspendedin 15 mL 0.2 micron filtered water and centrifuged to form a pellet andthe supernatant siphoned off. This was repeated 3 more times withfiltered water then repeated 3 more times with sterile (autoclaved)water. The pellet of Protein A-silica media was suspended in sterile(autoclaved) water to give a suspension volume of 8 mL. To each ofseveral test solutions was transferred 0.25 mL of the suspended sorbentrepresenting approximately 0.1 mL of packed Protein A-silica mediasorbent. Other tubes received only 0.25 mL of sterile water. Tubes werethen “spiked” with a microbial culture and left at room temperature forvarious periods of time whereupon a sample was plated out for countinglive cells as described above. Both E. coli and C. albicans were killedto less than 1% of the starting count while A. niger required up to 90hours for 100% killing. However, there was no detectable differencebetween killing rates in the presence or absence of the sorbent.

Example 4 Antimicrobial Effect vs. Time

An antimicrobial solution was made by titrating 10 mM acetic acid withNaOH to pH 6 then adding NaCl to 25 mM and benzyl alcohol and ethanoleach to 2% as above. Live cultures were added as a “spike” andperiodically live cells counted. After storing the solution for month,the microbial killing potential was still the same when tested with S.aureus.

Example 5 Antimicrobial Solution Stability

A solution of 10 mM acetic acid-NaOH-25 mM NaCl-2% benzyl alcohol-2%ethanol, pH 5.5, was stored for 22 months in high density polyethylenebottles with no detectable loss (less than 1% of its originalconcentration) of benzyl alcohol based on absorbancies measured at 257nm in water-diluted solutions.

Example 6 Antimicrobial Solution Stability in Presence ofChromatographic Media

Solutions of 2% each of benzyl alcohol and ethanol were made in 25 mMsodium phosphate, dibasic, and titrated to pH 6 with acetic acid.Solutions were kept at room temperature and at 5-8° C. for 4 months inpolypropylene bottles and tested for benzyl alcohol concentration andfor bactericidal capacity with E. coli using the antimicrobial effectdetermination by colony forming unit method. There was no detectableloss of benzyl alcohol nor loss of bactericidal capacity in the 4months.

Example 7 Stability of Chromatographic Media Stored in AntimicrobialSolutions

Bakerbond™ XWP 500 PolyABx-35 (500 Å) (#7586-02, Lot A24804)chromatographic media was rinsed several times with 100 mM sodiumacetate-acetic acid, pH 5, containing 2% benzyl alcohol and 2% ethanolthen suspended in an equal volume of the same benzylalcohol-ethanol-buffer combination. The suspension was left at roomtemperature for 2 years. The benzyl alcohol lost less than 2% of itsoriginal concentration based on absorbance assays and was found to havelost no detectable bactericidal potency when assayed by theantimicrobial effect determination by colony forming unit method. Thesorbent was periodically tested for capacity and resolution of testproteins and found to demonstrate no significant changes for up to 2years. The capacity of PolyABX-35 was measured by breakthrough methodusing rabbit gamma globulin. At the same conditions the 10% breakthroughcapacity at zero months was 54.5 mg/ml of resin and after twenty fourmonths was 54 mg/ml indicating no significant change in breakthroughcapacity.

Example 8 Stability of Affinity Chromatographic Media Stored inAntimicrobial Solutions

Protein A bonded silica chromatographic media was rinsed several timeswith 100 mM sodium acetate-acetic acid, pH 6, containing 2% benzylalcohol and 2% ethanol then suspended in an equal volume of the samebenzyl alcohol-ethanol-buffer combination. The suspension was left at4-8° C. for 4 months. The benzyl alcohol lost less than 2% of itsoriginal concentration based on absorbance assays and was found to havelost no detectable bactericidal potency when assayed by theantimicrobial effect determination by colony forming unit method.Further, the affinity media lost ligand from the resin to the liquidmedia at a much slower rate at pH 6 and higher than the rate at pH'slower than 6. The ligand leakage was measured with an enzyme-linkedimmunospecific assay (ELISA). The Leakage of Protein A detected by ELISAassays into the media at pH 5.0 was 2-3 times greater than at pH 6.0 andnearly 15 times greater at pH 4.5 than at pH 6.0.

While the invention has been described herein with reference to thespecific embodiments thereof, it will be appreciated that changes,modification and variations can be made without departing from thespirit and scope of the inventive concept disclosed herein. Accordingly,it is intended to embrace all such changes, modification and variationsthat fall with the spirit and scope of the appended claims.

1. A buffered antimicrobial solution for storage of chromatographicsolids, wherein the buffered anti-microbial solution comprises fromabout 1.5% to about 4% by weight benzyl alcohol, about 0.5% to about 4%by weight ethyl alcohol and from about 92% to about 98% by weight ofabout 50 mM to about 200 mM buffer to provide a solution at pH of about5.5 to about 7.5.
 2. A buffered antimicrobial solution according toclaim 1 wherein the pH is about pH 6.0.
 3. A buffered antimicrobialsolution according to claim 1 wherein the amount of benzyl alcohol isabout 2% by weight and the amount of ethyl alcohol is about 2% byweight.
 4. A buffered antimicrobial solution according to claim 1wherein the buffer is selected from the group consisting of sodiumcitrate, sodium acetate, sodium phosphate, triethanolamine, TRIS(tris(hydromethyl)aminomethane), HEPES(N-(2-hydroxyethyl)piperizine-N′-(2-ethanesulphonate), and MES(4-morpholineethanesulphonate).
 5. A buffered antimicrobial solutionaccording to claim 1 comprising about 2% by weight benzyl alcohol, about2% by weight ethanol and about 96% by weight 100 mM sodium phosphatedibasic titrated to pH 6.0.
 6. A method of storing a chromatographicsolid while preventing microbial infection of the solid comprisingretaining the chromatographic solid in a buffered antimicrobial solutionof claim
 1. 7. A method of storing a chromatographic solid whilepreventing microbial infection of the solid comprising retaining thechromatographic solid in a buffered antimicrobial solution of claim 2.8. A method of storing a chromatographic solid while preventingmicrobial infection of the solid comprising retaining thechromatographic solid in a buffered antimicrobial solution of claim 3.9. A method of storing a chromatographic solid while preventingmicrobial infection of the solid comprising retaining thechromatographic solid in a buffered antimicrobial solution of claim 4.10. A method of storing a chromatographic solid while preventingmicrobial infection of the solid comprising retaining thechromatographic solid in a buffered antimicrobial solution of claim 5.11. A method according to claim 6 wherein the chromatographic solid isselected from chromatographic media or chromatographic equipment.
 12. Amethod according to claim 11 wherein the chromatographic solid ischromatographic media.
 13. A method according to claim 6 wherein thechromatographic solid is stored in the antimicrobial solution for aperiod of up to about two years.
 14. A method according to claim 10wherein the chromatographic solid is stored in the bufferedantimicrobial solution for a period of up to about two years.
 15. Amethod according to claim 13 wherein the chromatographic solid ischromatographic media.
 16. A method according to claim 14 wherein thechromatographic solid is chromatographic media.
 17. A chromatographicsolid stored in a buffered antimicrobial solution, wherein the bufferedantimicrobial solution comprises from about 1.5% to about 4% by weightbenzyl alcohol, about 0.5% to about 4% by weight ethyl alcohol and fromabout 92% to about 98% by weight of about 50 mM to about 200 mM bufferto provide a solution at pH of about 5.5 to about 7.5.
 18. Achromatographic solid according to claim 17 wherein the chromatographicsolid is selected from chromatographic media or chromatographicequipment.
 19. A chromatographic solid according to claim 17 wherein thechromatographic solid is stored in the buffered antimicrobial solutionfor a period of up to about two years.
 20. A chromatographic solidaccording to claim 17 wherein the buffered antimicrobial solutioncomprises about 2% by weight benzyl alcohol, about 2% by weight ethanoland about 96% by weight 100 mM sodium phosphate dibasic titrated to pH6.0.